New methodology is proposed for the in vivo labeling of recombinant proteins with small-molecule probes (e.g., fluorophores, crosslinkers, photo switches, and spin labels). This methodology will employ re-engineered mutants of biotin ligase (BirA), a bacterial enzyme that site-specifically biotinylates a lysine side-chain within a 13-amino acid consensus sequence (the "acceptor peptide"; modified lysine residue shown in blue, below). By altering the biotin binding site of BirA to accommodate a range of biotin analogues and other small molecules, we expect to generate enzymes ("probe ligases") that site-specifically conjugate various probes to recombinant proteins bearing the acceptor peptide. Protein-conjugated biotin analogues beating ketone, azide, or alkyne groups can be bio-orthogonally ligated to appropriately functionalized fluorophores or other biophysical probes via hydrazone formation, Staudinger ligation, and [3+2] Sharpless cycloaddition, respectively. Specific aims: 1. Development of a probe ligase that works in vitro. 2. Development of a probe ligase that works in living cells. 3. Application of the methodology to identification of protein interaction partners for glutamate receptor.